before and after the onset of cell death and replacement fibrosis. Mice with cardiac overexpression of wild-type
Dsg2 and wild-type mice served as controls. Assessment by electron microscopy established that intercellular
space widening at the desmosomes/adherens junctions occurred in Tg-NS/L mice before the onset of necrosis
and fibrosis. At this stage, epicardial mapping in Langendorff-perfused hearts demonstrated prolonged ventricular ac-
tivation time, reduced longitudinal and transversal conduction velocities, and increased arrhythmia inducibility. A
reduced action potential (AP) upstroke velocity due to a lower Na+current density was also observed at this
stage of the disease. Furthermore, co-immunoprecipitation demonstrated an in vivo interaction between Dsg2 and
the Na+channel protein NaV1.5.
Intercellular space widening at the level of the intercalated disc (desmosomes/adherens junctions) and a concomitant
reduction in AP upstroke velocity as a consequence of lower Na+current density lead to slowed conduction and
increased arrhythmia susceptibility at disease stages preceding the onset of necrosis and replacement fibrosis. The
demonstration of an in vivo interaction between Dsg2 and NaV1.5 provides a molecular pathway for the observed
electrical disturbances during the early ARVC stages.
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Arrhythmias † Sudden cardiac death † Electrophysiology † ARVC † Desmoglein 2
Intercalated disc abnormalities, reduced
Na1current density, and conduction slowing
in desmoglein-2 mutant mice prior
to cardiomyopathic changes
Stefania Rizzo1†, Elisabeth M. Lodder2†, Arie O. Verkerk3, Rianne Wolswinkel2,
Leander Beekman2, Kalliopi Pilichou1, Cristina Basso1, Carol Ann Remme2,
Gaetano Thiene1, and Connie R. Bezzina2*
1Department of Cardiac, Thoracic and Vascular Sciences, University of Padua, Padua 35121, Italy;2Department of Experimental Cardiology, Heart Failure Research Center, Academic
Medical Center, University of Amsterdam, Room L2-108-1, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands; and3Department of Anatomy, Embryology and Physiology, Heart
Failure Research Center, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
Received 6 June 2012; revised 19 June 2012; accepted 28 June 2012
Mutations in genes encoding desmosomal proteins have been implicated in the pathogenesis of arrhythmogenic right
ventricular cardiomyopathy (ARVC). However, the consequences of these mutations in early disease stages are
unknown. We investigated whether mutation-induced intercalated disc remodelling impacts on electrophysiological
properties before the onset of cell death and replacement fibrosis.
Transgenic mice with cardiac overexpression of mutant Desmoglein2 (Dsg2) Dsg2-N271S (Tg-NS/L) were studied
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an im-
portant cause of ventricular arrhythmias and sudden cardiac death, es-
pecially in the young and in athletes.1Mutations in one or more genes
encoding desmosomal proteins are found in ?50% of patients.2–6
Although uncovering the genetic basis of ARVC provided us with mo-
lecular leads to investigate the pathogenesis of the disease,7the
mechanisms of the early cardiac electrical instability in ARVC are
Desmosomes are highly conserved structures that, together with
adherens junctions and gap junctions, connect cardiac myocytes end
†Both authors contributed equally to this work.
* Corresponding author. Tel: +31 20 5665403; fax: +31 20 6976177, Email: email@example.com
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2012. For permissions please email: firstname.lastname@example.org.
Cardiovascular Research Advance Access published July 31, 2012
by guest on January 28, 2015
to end at the level of the intercalated discs (ID) and thereby play a
crucial role in maintaining proper myocardial function.8Altered
desmosomal organization, as the result of mutations, is thought to
lead to myocardial damage and replacement fibrosis, the classical
histopathologic pattern of ARVC.1In advanced stages of the
disease, focal scars cause electrical isolation of cardiomyocytes
within non-conducting fibrous tissue, resulting in slow conduction
and delayed activation, thus forming the substrate for re-entrant
circuits and ventricular electrical instability.
However, arrhythmias have also been described early in the disease
process,9before overt structural changes of the myocardium.10This
phenomenon may be due to abnormalities in electrical coupling
between cardiomyocytes that are already present before the onset
of cardiomyopathic changes. Recent findings indicating the presence
of mixed-type junctions (the area composita)11and crosstalk
between protein complexes pertaining to the different types of junc-
tions12–17have markedly changed the perception of the ID. These
data support the concept of cross-regulation between structural
and electrical components at the ID.
In this study, we employed our ARVC mouse model with overex-
pression of mutant Desmoglein-2 (Dsg2-N271S).7This mutation is the
homologue of the DSG2-N266S mutation identified in an ARVC
patient from Padua.5A total of 45 mutations in DSG2, mostly affecting
the adhesive extracellular domains of Dsg2, have been reported thus
far,18accounting for approximately 8% of the mutations associated
with human ARVC. We used this model to investigate whether ID re-
modelling, as a consequence of a mutation in a component of the
desmosome, impacts on cardiac electrophysiological properties at
early disease stages that is before the onset of fibrosis and other car-
diomyopathic changes. We show that mutant Dsg2 induces widening
of the ID at the level of the area composita. This coincides with con-
duction slowing stemming from a reduced action potential (AP) up-
stroke velocity caused by a reduction in the cardiac Na+current
(INa) density. Furthermore, we demonstrate that Dsg2 and the
cardiac Na+channel (NaV1.5) interact in vivo, pointing to a molecular
mechanism for the development of conduction slowing and arrhyth-
mia in ARVC prior to gross and histological changes of the heart.
2.1 Animal husbandry and mouse lines used
We studied the effects of heart-specific overexpression of mutant Dsg2 in
young mice before the development of gross and histological abnormal-
ities. The transgenic mouse models with heart-specific overexpression
of either mutant (Dsg2-N271S) or wild-type (Tg-WT) Dsg2 were gener-
ated previously.7Of the two Dsg2-N271S lines we generated, which re-
spectively had high (Tg-NS/H) and low (Tg-NS/L) overexpression of the
transgene, the Tg-NS/L line was used in this study. Although both lines,
i.e. Tg-NS/H and Tg-NS/L, develop an ARVC phenotype, the latter line
was selected as it develops the phenotype at a slower pace which
makes it more amenable for the dissection of the early, pre-
cardiomyopathic, phenotype of the model. Tg-WT and wild-type (WT)
mice were used as controls in all experiments throughout the study.
The mice were studied at three different ages: ,2 weeks, 3–4 weeks,
and 6–9 weeks. At least four mice of each group were studied except
where specifically indicated otherwise. Mice were sacrificed by cervical
dislocation after sedation with O2/CO2for 1 min. All experiments were
approved by the local animal welfare committee and followed Dutch
law concerning experimental animal welfare and conformed with the
Guide for the Care and Use of Laboratory Animals published by the
US National Institutes of Health (NIH Publication No. 85-23, revised
1996) (PHS assurance number A5549-01).
2.2 Morphological analysis
For pathological studies, hearts were isolated and snap-frozen immediate-
ly after excision in liquid nitrogen and stored at 2808C. In parallel, tissue
samples were fixed in 4% paraformaldehyde (in PBS) for light microscopy
or in glutaraldehyde for transmission electron microscopy (TEM) (see
below). Seven-mm-thick paraffin embedded sections were cut and rou-
tinely stained with haematoxylin and eosin (H&E) and Heidenhain’s tri-
chrome to examine the myocardium and to detect the presence and
amount of necrosis, inflammation, and fibrosis.
2.3 Transmission electron microscopy
Transmission electron microscopy was used to characterize the desmo-
somes and the other intercellular junctions in situ as described before.11
Electron micrographs were taken by systematic random sampling and ana-
lysed by two independent expert pathologists (S.R. and C.B.) blinded to
the genotype of the mouse. For evaluation, ?100 IDs were analysed
per mouse. Morphometric analysis of ID space was performed according
to a previously described method.19We also calculated the percentage of
junctions with an intercellular space . 30 nm.20
2.4 Electrical analysis
The electrical properties of the heart were studied in vivo by surface elec-
trocardiograms (ECG), ex vivo in Langendorff-perfused hearts by epicardial
mapping and in isolated cardiomyocytes by patch-clamp analysis. Shortly:
epicardial mapping: ventricular extracellular epicardial electrograms were
recorded from the right ventricle (RV) and left ventricle (LV). Maximal
conduction velocities in both longitudinal and transverse directions
were measured from RV and LV activation maps. Cellular electrophysiology:
mouse ventricular myocytes were isolated by enzymatic dissociation as
described previously in detail.21APs and INawere recorded with the
amphotericin-B-perforated patch-clamp and ruptured patch-clamp tech-
nique, respectively. Current-clamp experiments: APs were measured at
368C and data from 10 consecutive APs were averaged. Voltage-clamp
experiments: INawas measured using a two-step protocol at room tem-
perature (RT), with a holding potential of 2120 mV and a cycle time of
5 s (Figure 6D). Details of all electrical experiments are given in Supple-
mentary material online.
2.5 Immunofluorescence microscopy
Cryosections of 5 mm were fixed in methanol (5 min) followed by
acetone (20 s), at 2208C, air-dried, and rehydrated in PBS. Permeabil-
ization was done in 0.2% Triton X-100 for 5 min. Primary antibodies
were applied for 1 h at RT, followed by three washes in PBS
(5 min each), incubation with the secondary antibodies (30 min, RT),
and 3 × 5 min washes with PBS. Sections were mounted with 50%
glycerol in PBS. Images were recorded with a confocal laser scanning
microscopy (Leica CTR 5500). Details of the used antibodies are given
in the Supplementary material online.
2.6 Protein isolation and western blot analysis
Proteins were isolated from LV tissue from a snap frozen mouse heart and
western blotting was performed according to the standard procedures. In
short: LV protein (60 mg) was run on denaturing SDS-page gels. The
gels were blotted on a pre-equilibrated PVDF Immobilon-P membrane
(Millipore) by means of a semidry system. Blots were cut at appropriate
heights and probed with primary antibodies. HRP conjugated secondary
antibodies were detected with ECL-Plus (Amersham). Chemiluminescent
signals were visualized using a digital image analyzer (LAS-4000 Lite;
Fujifilm) and quantified using the Aida software package (Aida Image
S. Rizzo et al.
Page 2 of 10
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Analyzer v.4.26). Details of the used antibodies and protein isolation are
given in the Supplementary material online.
Aliquots of 100 mg LV whole cell lysate protein of 3–4-week-old mice
were incubated rotating overnight at 48C with washed agarose beads
with either conjugated M2 Flag antibody or protein A (both Sigma) and
1 mL of normal mouse IgG (Santa Cruz) in 1 mL of PBS supplemented
with protease inhibitors (Complete Mini; Roche) and 0.5 mM Sodium
Orthovanadate (PBS++). Beads were washed three times with 1 mL of
PBS++, and the bound proteins were eluted with Flag peptide (Sigma).
Untreated protein and elutes from both precipitations were treated
with Laemli buffer and analysed by western blot as described above.
2.8 Surface ECGs
Mice were anaesthetized using isoflurane inhalation (0.8–1.0% volume in
oxygen), and efficacy of the anaesthesia was monitored by watching
breathing speed and tail suspension. Four-lead surface ECGs were
recorded from subcutaneous 23-gauge needle electrodes attached to
each limb using the Powerlab acquisition system (ADInstruments). Lead
II was analysed for heart rate (RR interval) and PR, QRS, and QT duration
using Chart5 Pro analysis software (ADInstruments). QT intervals in mice
were corrected for heart rate using the following formula: QTc ¼ QT/
(RR/100)1/2 (RR in ms).
Detailed descriptions of morphological analysis, TEM, antibodies, im-
munofluorescence microscopy, protein isolation, western blot analysis,
co-immunoprecipitation, surface ECG, epicardial mapping experiments,
cellular electrophysiology, and statistical analysis are presented in the Sup-
plementary material online.
2.9 Statistical analysis
Data are expressed as mean+SEM. Values are considered significantly
different if P , 0.05 in an unpaired two-sided t-test or in two-way
repeated measures of analysis of variance (two-way repeated measures
ANOVA) followed by pairwise two-sided comparison using the
Student–Newman–Keuls test, after testing for normal distribution of
the data. Minimal sample sizes were calculated prior to the experiments
based upon a power calculation with a (12b) ¼ 80%, a ¼ 5%, mean
control ¼ 7.0, mean observed ¼ 9.5, and SD ¼ 1.0 (effect size of 2.5,
which represents the expected effect sizes for the electrical mapping)
which gives n ¼ 4 animals per test group. The intraclass correlation coef-
ficient was used to test the interobserver variability in the morphometric
measurements. Statistical tests were performed using the PASW statistics
software, version 18.0.2 (IBM, New York, NY, USA) and SigmaStat,
version 3.1 (Aspire Software International, Ashburn, VA, USA).
3.1 Absence of cardiomyopathic changes in
Tg-NS/L mice younger than 6 weeks of age
The early phase of the disease was studied by investigating mice at
three different age groups: (i) ,2 weeks of age, (ii) 3–4 weeks of
age, and (iii) 6–9 weeks. On gross examination and histologically,
hearts from Tg-NS/L mice at all three age groups appeared normal,
with no evidence of replacement-type fibrosis (Supplementary mater-
ial online, Figure S1). Other cardiomyopathic changes, consistent with
those we previously reported for Tg-NS/H mice7and which included
necrosis, focal myofibrillar lysis, dilated sarcoplasmatic reticulum and
T-tubules, and mitochondrial clustering, were observed exclusively
in Tg-NS/L mice ≥6 weeks of age (Figure 1). These analyses estab-
lished that Tg-NS/L mice ,6 weeks of age were devoid of
cardiomyopathic changes allowing for the examination of the early
electrophysiological phenotype prior to and in the absence of
3.2 Widening of intercellular space
at the desmosome/adherens junctions
We next examined ID structures in detail by TEM. No consistent dif-
ferences were observed in the general organization of the cell–cell
junctions between Tg-NS/L and control mice. Gap junctions appeared
structurally normal in all groups (Figure 1). In addition to desmosomes,
gap junctions, and adherens junctions, intermediate structures were
observed that displayed features of both desmosomal and adherens
junctions concurring with recent reports describing this ‘area
Separation of the opposed membranes, resulting in larger intercel-
lular spaces, was observed at the level of desmosomes/adherens junc-
tions. These changes were seen in otherwise morphologically normal
cardiomyocytes in all Tg-NS/L mice ≥3 weeks of age and in 1 out of 4
Tg-NS/L mice ,2 weeks old (Figure 1). None of the control mice
(Tg-WT, WT) displayed any intercellular space widening. Morpho-
metric analysis showed that the average intercellular space was signifi-
cantly widened in Tg-NS/L mice at 3–4 weeks compared with
controls (Supplementary material online, Figure S2A). The percentage
of widened cellular junctions increased with age (Supplementary ma-
terial online, Figure S2B). In some Tg-NS/L cardiomyocytes with inter-
cellular space widening, myofibrils appeared to have undergone focal
lysis at their points of attachment to desmosomes/adherens junctions
3.3 Conduction slowing in Tg-NS/L hearts
from 3 to 4 weeks of age
To investigate the occurrence of electrophysiological abnormalities
prior to the development of cardiomyopathic changes, we first per-
formed surface ECG measurements in anaesthetized mice. In mice
aged 3–4 weeks, no statistically significant differences in heart rate
(RR interval), QRS duration, PR interval, and QTc interval were
observed between Tg-NS/L and controls (Figure 2A and B and Supple-
mentary material online, Table S1). However, fractionation of the QRS
complex was observed in some Tg-NS/L mice aged 3–4 weeks, indi-
cating the presence of discrete ventricular conduction slowing
(Figure 2A). In the 6–9 weeks age group, Tg-NS/L mice developed sig-
nificant QRS prolongation and abnormal QRS morphology (including
marked fractionation), in addition to spontaneous ventricular rhythm
disturbances consisting of single or multiple ventricular extra systoles
and short runs of non-sustained ventricular tachycardia (Figure 2C).
No spontaneous arrhythmias were observed in Tg-NS/L mice aged
We next performed epicardial mapping in isolated Langendorff-
perfused hearts from WT, Tg-WT, and Tg-NS/L mice of the three
age groups to further assess cardiac conduction in detail. In mice
aged ,2 weeks, no significant differences were found between the
groups for LV or RV activation time or effective refractory periods,
but a tendency towards lower longitudinal and transversal conduction
velocities was observed in Tg-NS/L mice compared with controls
(Figure 3A–D, Supplementary material online, Table S2). In contrast,
a significantly prolonged epicardial LV activation time in addition to
decreased LV conduction velocity was observed in Tg-NS/L mice
from the age of 3–4 weeks onwards compared with controls. Both
Early changes in mutant Dsg2 mice
Page 3 of 10
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longitudinal and transversal conduction velocities were equally
affected, as indicated by the unaltered L/T ratio (Supplementary ma-
terial online, Table S2). In the RV, activation time and conduction vel-
ocity were only significantly prolonged at the age of .6 weeks.
Arrhythmia inducibility was tested using up to three extra stimuli
and burst pacing. No arrhythmias could be induced in WT, Tg-WT,
or Tg-NS/L mice younger than 2 weeks. In contrast, ventricular
arrhythmias were inducible in almost half of all Tg-NS/L mice aged
3–4 weeks and .6 weeks, but only sporadically in control mice of
the same age groups (Supplementary material online, Table S2).
3.4 Localization and levels of the
intercalated disc proteins
We hypothesized that the conduction slowing observed in hearts at
3–4 weeks of age, i.e. prior to the onset of cardiomyopathic
Figure 1 At the ultrastructural level, widening of the intercellular space at the level of the desmosomes/adherens junctions compared with WT
mice (A) and Tg-WT (B) is visible in Tg-NS/L mice both at 3–4 weeks (D and E) and at 6–9 weeks (F). This feature is observed also in a Tg-NS/
L mouse aged ,2 weeks (C). The structure of the gap-junctions is preserved in both Tg-NS/L and Tg-WT mice at all ages (B–F). Scale bars 500 nm.
S. Rizzo et al.
Page 4 of 10
by guest on January 28, 2015
changes, could be related to altered localization or reduced levels of
components of the ID. We used immunofluorescence to characterize
the distribution of ID proteins in Tg-NS/L mice and control mice. A
Desmoglein-2, Desmocollin-2, Plakophilin-2 (Pkp2), Plakoglobin
(PG), Desmoplakin, and NaV1.5 as well as for the classical adherens
junction proteins N- and pan-Cadherin and a- and b-Catenin and
the gap junctional Connexin43 (Cx43) (Figure 4A, Supplementary ma-
terial online, Figure S3, and data not shown). Note the normal
co-localization of Cx43 with pan-Cadherin in all three genotype
groups; no clear lateralization of Cx43 was observed (Figure 4A).
On western blot analysis in 3–4-week-old hearts, no differences
were observed in the levels of PG, Pkp2, NaV1.5, Pan-Cadherin, and
Cx45 between the three genotype groups (Figure 4B and Supplemen-
tary material online, Figure S4). However, we observed a significantly
reduced level of Cx43 in Tg-WT compared with WT hearts (Figure 4B
and C). Additionally, we observed a shift in the height of the Cx43
bands from predominantly phosphorylation state P2 as previously
described22in WT to P1 and P0 in both Tg-WT and Tg-NS/L lines
was detected forFlag-tagged
3.5 Reduced AP upstroke velocity
in isolated cardiomyocytes
In parallel, we investigated the possible cellular electrophysiological
changes underlying the ventricular conduction slowing in the
cardiomyocytes isolated from 3 to 4-week-old mice were measured
using patch clamp methodology. Figure 5A shows typical APs of
Tg-WT and Tg-NS/L myocytes; Figure 5B summarizes the average AP
characteristics of WT, Tg-WT, and Tg-NS/L myocytes. No differences
were noted between Tg-NS/L, Tg-WT, and WT in resting membrane
potential (RMP), AP amplitude (APA), and AP duration (APD) at 20,
50, and 90% repolarization (APD20, APD50, and APD90, respectively).
However, cardiomyocytes from Tg-NS/L mice showed a significantly
lower AP upstroke velocity (Vmax) compared with age-matched WT
and Tg-WT mice (Figure 5B). On average, Vmaxin Tg-NS/L myocyte
mined by Na+influx through voltage-gated Na+channels. The
observed lower Vmaxthus indicates that cardiomyocytes of Tg-NS/L
mice have a reduced functional Na+channel availability.21
The reduced functional Na+channel availability during the AP up-
stroke may be due to a decrease of INadensity and/or changes in
voltage-dependency of (in)activation. Voltage-clamp experiments per-
formed at RT using a double pulse protocol (Figure 6D) demonstrated
no differences in the voltage dependencies of the activation (Figure 6A)
and inactivation (Figure 6B) of INa. Figure 6C shows the current–
voltage relationships of INain WT, Tg-WT, and Tg-NS/L myocytes.
Maximal peak currents were smaller in Tg-NS/L myocytes compared
with WT and Tg-WT myocytes (Figure 6D). On average, the maximal
peak current was ≈19+6% lower; thus, the reduction in INadensity
was in the same order as the Vmaxreduction.
Figure 2 (A) Surface ECG examples of WT, Tg-WT, and Tg-NS/L mice (scale bar: 20 ms). In a subset of Tg-NS/L mice aged 3–4 weeks, discrete
fractionation of the QRS complex was observed (top right example). In the 6–9 weeks age group, Tg-NS/L mice developed significant QRS prolonga-
tion and abnormal QRS morphology (including substantial fractionation of the QRS complex). (B) Average values (mean+SEM) for QRS duration in
WT, Tg-WT, and Tg-NS/L mice in age groups 3–4 weeks (n ¼ 7, 7, and 12, respectively) and 6–9 weeks (n ¼ 10, 9, 12, respectively) (# denotes
P , 0.05 vs. WT; $ denotes P , 0.05 vs. Tg-WT). Mean values for all ECG parameters are presented in Supplementary material online, Table S1.
(C) Examples of spontaneous ventricular rhythm disturbances observed in Tg-NS/L mice aged 6–9 weeks, including single or multiple ventricular
extra systoles and short runs of non-sustained ventricular tachycardia (scale bar: 100 ms).
Early changes in mutant Dsg2 mice
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Figure 3 (A) Typical examples of LV activation maps of isolated Langendorff-perfused hearts from WT, Tg-WT, and Tg-NS/L mice aged 3–4 weeks.
Arrows indicate directions and distances used for measurements of longitudinal (CV-L) and transversal (CV-T) conduction velocities. Crowding of
isochrones (1 ms) in the Tg-NS/L heart indicates areas of conduction slowing. (B) Average values (mean+SEM) for LV and RV total activation
time. (C and D) Average values (mean+SEM) for LV and RV longitudinal and transversal conduction velocities (B–D) in hearts from WT,
Tg-WT, and Tg-NS/L mice aged ,2 weeks (n ¼ 6, 4, 5, respectively), 3–4 weeks (n ¼ 5, 6, 6, respectively), and .6 weeks (n ¼ 6, 5, 6, respectively)
(* denotes P , 0.05 vs. WT; $ denotes P , 0.05 vs. Tg-WT). Average values for all parameters and ANOVA P-values are presented in Supplementary
material online, Table S2. (E) Examples of ventricular arrhythmias induced in isolated Langendorff-perfused hearts of Tg-NS/L mice aged 3–4 weeks
(scale bar: 100 ms). Top panels show induction of non-sustained polymorphic ventricular tachycardia induced by either three extrasimuli (top left
panel) or burst pacing (top right panel). The lower panel depicts an example of a sustained monomorphic ventricular tachycardia induced
through burst pacing.
S. Rizzo et al.
Page 6 of 10
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3.6 Physical interaction between Dsg2
Since our electrophysiological studies showed a reduced INadensity in
Tg-NS/L mice, and, considering the fact that recent studies in rat neo-
natal cardiomyocytes have demonstrated that desmosomal proteins
tigate the possible in vivo interaction between Dsg2 and NaV1.5 by
co-immunoprecipitation. We made use of the Flag-tag epitope
present on the Dsg2 protein overexpressed in Tg-WT and Tg-NS/L
mice to efficiently and specifically pull down Dsg2 from whole cell
protein extracts of the LV of transgenic mice. This demonstrated an
interaction between Flag-tagged Dsg2 and NaV1.5 in both Tg-WT
and Tg-NS/L hearts (Figure 7).
We here demonstrate for the first time that a mutation in a structural
component of cardiac desmosomes impacts on ventricular conduction
and arrhythmia susceptibility even before the onset of necrosis and re-
placement fibrosis. These effects occur through a reduction in AP up-
stroke velocity due to a reduced INadensity. Furthermore, we show
for the first time that the cardiac Na+channel in an in vivo murine
model forms part of a macromolecular complex that includes Dsg2.
This structural link between the desmosomal protein complex and
the NaV1.5 channel may explain the conduction disturbances and
arrhythmias seen early in the ARVC disease process.
Our findings provide support to the recent proposition that at the
ID, cross-talk exists between structures previously perceived as being
independent.14In a series of recent studies, the Delmar group demon-
strated interactions between Pkp2, Cx43, NaV1.5, and AnkG, thus
connecting the desmosomes to the gap junctions and the Na+
channel complex.12,23In these previous studies, disruption of these
protein complexes by downregulation of Pkp2 and/or AnkG in cul-
tured neonatal rat cardiomyocytes led to reduced Na+channel avail-
ability. The data presented in the current study now shows that Dsg2
interacts with NaV1.5 in the mouse heart in vivo. This observation
Figure 4 (A) Immunohistochemistry of Cx43 (green) and pan-Cadherin (Cdh, red) on LV tissue of WT, Tg-WT, and Tg-NS/L mice at 3–4 weeks.
(B) Western blot analysis of whole cell lysate of hearts of WT, Tg-WT, and Tg-NS/L mice aged 3–4 weeks for Calnexin, NaV1.5, and Cx43. (C) Quan-
tification of the NaV1.5 and Cx43 western blot signals (n ¼ 3) relative to Calnexin normalized for WT, error bars denote standard errors, and *
denotes P , 0.05 vs. WT.
Early changes in mutant Dsg2 mice
Page 7 of 10
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coupled to the reduced AP upstroke velocity due to reduced INa
density point to this functional link as the basis for the observed
reduction in conduction velocity. It will be interesting to study the
temporal and spatial changes in these interactions, as well as the inter-
action with the other molecules in this protein complex; these studies
are unfortunately impossible with co-immunoprecipitation, but will
need real-time in vivo imaging of the components of ID.
Unexpectedly, Cx43 protein levels were found to be significantly
reduced in Tg-WT; furthermore, a shift towards less-phosphorylated
(P0, P1) forms of Cx43 was observed. Nevertheless, Cx43 sub-
cellular localization and distribution within the myocardium did not
appear to be altered compared with WT mice, in particular no
clear lateralization, which has been reported to correlate to the phos-
hporylation state of Cx4322, was observed. Previously, homogeneous
reduction in Cx43 has been shown to be well tolerated. For instance,
no differences in cardiac conduction were observed in mice carrying a
heterozygous deletion of Cx43.25,26The observed reduction of ?25%
of Cx43 in both Tg-NS/L and Tg-WT mice is therefore not expected
to influence conduction parameters. Corroborating this, no differ-
ences on surface ECG and in epicardial mapping were observed
between Tg-WT and WT animals. However, we cannot exclude
the possibility that the observed reduction in Cx43 levels sensitizes
the hearts of the Tg-NS/L mice to the consequences of the Dsg2 mu-
tation. The fact that no other changes in level and localization of ID
proteins were seen in Tg-NS/L mice at 3–4 weeks indicates that
the observed changes in conduction are due to subtle changes of
protein–protein interactions of the ID proteins rather than gross
changes in their level and localization.
tion with focal lysis of the myofilaments, as previously described in
humans.19In our previous studies on Tg-NS/H mice,7which have a
faster disease development, the widening of the intercellular space
wasdetected only inthe setting ofconcomitant necrosis and inflamma-
tion and as such was interpreted as a secondary phenomenon. In the
current study, the use of the Tg-NS/L line, which is characterized by a
later disease onset, allowed us to show that ID space widening is an
early feature of the disease and precedes cell injury and inflammation.
Such ID widening could be explained by the fact that the N271
residue, located between the second and third extracellular cadherin
domains of Dsg2, has been shown to be critical for co-ordination of
Ca2+binding, a phenomenon essential to the adhesive intercellular
interactions of junctional cadherins.27Similar observations were also
made by Kant et al.28who studied mice carrying a deletion of the adhe-
sive extracellular domain of Dsg2. They suggested that mutant Dsg2
results in compromised adhesion at ID and mechanical cell stress
death, inflammation, and fibrotic replacement. These features are
similar to those observed in our Tg-NS/L mouse model, showing an
age-related development of structural lesions, although in the current
investigation we focused on the early stages when the heart is still
grossly and histologically normal.
In 3–4-week-old mice, intercellular space widening coincided with
the onset of conduction slowing. Here, two scenarios are possible. In
one, desmosomal interactions27are weakened by the N271S muta-
tion leading to intercellular space widening, consequently disrupting
Figure 5 (A) Representative APs of LV cardiomyocytes isolated from a Tg-NS/L (light grey) and Tg-WT (black) mouse heart of 3–4 weeks. Inset:
First derivatives of the AP upstrokes. (B) Average AP characteristics of Tg-NS/L (n ¼ 3, n ¼ 11), Tg-WT (n ¼ 3, n ¼ 13), and WT (n ¼ 3, n ¼ 11)
cardiomyocytes aged 3–4 weeks. RMP, resting membrane potential; APA, maximal AP amplitude; Vmax, maximal upstroke velocity; APD20, APD50
and, APD90¼ AP duration at 20, 50, and 90% repolarization, respectively.
S. Rizzo et al.
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the multiprotein Na+channel complex. In a second scenario, the Dsg2
mutation leads to a conformational change affecting the functional
interaction between the desmosomal complex and the Na+channel
complex independent of the intercellular space widening.
The conduction slowing and increased arrhythmia inducibility in the
3–4-week-old Tg-NS/L mice point to electrical instability prior to
overt structural changes. However, no spontaneous arrhythmias
were observed at this age on surface ECG, although we cannot
exclude their occurrence since long-term telemetric ECG recordings
are not feasible at this young age. Clearly, the observed conduction
slowing likely sensitizes the Tg-NS/L mice for development of spon-
taneous arrhythmias at more advanced disease stages. Our experi-
mental findings of altered ventricular conduction and increased
arrhythmia susceptibility even before the onset of necrosis and re-
placement fibrosis support the hypothesis that conduction distur-
bances and electrical instability could develop in human carriers of
ARVC gene mutations without structural changes (pre-clinical phase
of ARVC). This finding underlines the need of a diagnostic tool
targeting conduction changes, especially in the cardiological screening
of first-degree relatives of ARVC probands carrying gene mutations.
Epicardial mapping in Tg-NS/L mice indicates that the LV is more
affected than the RV. This is in line with the increasing recognition
Figure 6 Na+current (INa) characteristics in WT, Tg-WT, and Tg-NS/L myocytes. (A) Voltage dependency of activation. (B) Voltage dependency of
inactivation. (C) Current-voltage relationships of INa. (D) Voltage clamp protocol. (E) Average maximal peak currents, * denotes P , 0.05 vs. WT.
Figure 7 Co-immunoprecipitation of NaV1.5 with Flag-Dsg2 in
Tg-WT and Tg-NS/L mice of 3–4 weeks: western blot for NaV1.5
and Flag tagged Dsg2 on whole cell lysate (In), whole cell lysate pre-
cipitated with normal mouse IgG (IgG, negative control), and whole
cell lysate precipitated with M2 a-Flag (M2).
Early changes in mutant Dsg2 mice
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of biventricular involvement in ARVC.1,5,9,29,30The relative rarity of Download full-text
the left-dominant involvement in published ARVC populations is
likely a consequence of restrictive inclusion criteria and low sensitivity
of diagnostic tools.31In the setting of family history of ARVC, even
signs of isolated LV involvement should be carefully investigated, as
they could be the only marker of the underlying genetically deter-
mined cardiomyopathy.9,30Noteworthy, all the reported experimen-
tal animal models irrespective of the affected gene show both LV and
In summary, we here dissect the early stages of disease develop-
ment in mice overexpressing a Dsg2 mutation associated with
ARVC in humans. Intercellular space widening at the level of the ID
(desmosomes/adherens junctions) and a concomitant reduction in
AP upstroke velocity as a consequence of reduced functional Na+
channel availability leads to slowed conduction and increased arrhyth-
mia susceptibility at disease stages preceding the onset of replacement
fibrosis. The demonstration of an in vivo interaction between Dsg2 and
NaV1.5 provides a molecular pathway for the observed electrical dis-
turbances during the early ARVC disease process.
Supplementary material is available at Cardiovascular Research online.
Conflict of interest: none declared.
This work was supported by the Netherlands Heart Foundation
(2009B051 and 2005T024), the Netherlands Heart Institute (ICIN,
061.02), and the Division for Earth and Life Sciences (ALW) of the Neth-
erlands Organization for Scientific Research (NWO) (836.09.003); the
Registry for Cardio-Cerebro-Vascular-Pathology, Veneto Region, Venice;
Pricard Conacuore, Modena; and the CARIPARO Foundation, Padua,
Italy. During this investigation, Dr S. Rizzo was a visiting researcher
from the University of Padua at the Academic Medical Center, University
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